Resorcinol and m-guaiacol alkylated derivatives and asymmetrical secondary alcohols in the leaves from Tamarix canariensis

Submitted to Phytochemistry letters Resorcinol and m-guaiacol alkylated derivatives and asymmetrical secondary alcohols in the leaves from Tamarix canariensis Josep Basas1, Jordi López2 and F. Xavier C. de las Heras1,* 1Department of Mining Engineering and Natural Resources – Escola Politècnica Superior d’Enginyeria de Manresa Universitat Politècnica de Catalunya (UPC) Av. Bases de Manresa 61-73 E-08242 Manresa, Catalonia, Spain 2Department of Environmental Chemistry IDAEA-CSIC, Jordi Girona 18-26, 08034 Barcelona, Catalonia, Spain *Corresponding author. Telf.: + 34 938 777 236; Fax: + 34 938 777 202; E-mail address: [email protected] Graphical abstract HO OH OCH 3 HO OH Abstract In the study of leaves from the halotolerant saltcedar (Tamarix canariensis Willd.) we have found a new abundant source of resorcinolic as well as guaiacolic compounds. A homologous series of 5-n-alkylresorcinols with long side alkyl chain reported previously in the literature almost exclusively in cereals are described here for the first time in Tamaricaceae. These polyketide derived phenolic compounds are characterized by a series ranging from n-C15 to n-C27 and maximizing at n-C21 including nonnegligible amounts of the even-numbered homologues. We also have characterized for the first time as natural compounds a homologous series of 5-n-alkylguaiacols similarly constituted by an odd-numbered alkyl chain linked to the benzene ring at position 5. They also include a hydroxyl group at carbon 1 of the phenolic nucleus and a methoxy group at position 3. The homologues are in the range of 13-27 carbon atoms (Cmax at C21). Finally, we firstly report in Tamaricaceae eight series of unsymmetrical secondary alcohols ranging from n-C25 to n-C35 with hentriacontan-12-ol as the major homologue. This saltcedar leaves waxes contained high amounts of alkylresorcinol (17,2 g/kg dw), alkylguaiacol (14,4 g/kg dw) and secondary alcohols (44,4 g/kg dw). 1 Highlights: -Alkylguaiacols described for the first time as natural products. -We characterize the mass spectrum of their trimethylsilylderivatives never shown. -First report on secondary alcohols and alkylresorcinols in Tamaricaceae. Keywords: Tamarix canariensis Willd.; saltcedar tree; Tamaricaceae; Leaf epicuticular waxes; alkylresorcinols; alkylguaiacols; secondary alcohols; total extracts; Gas chromatography-Mass spectrometry (GC/MS); trimethylsilyl (TMS) derivatives. 1. Introduction The saltcedar tree (canary island tamarisk) (Tamarix canariensis Willd.) is a perennial shrub to small tree member belonging to the Tamaricaceae family that growths in saline and temporally flooded soils. This macrophanerophyte member of the riparian and halophytic vegetation is native to Northern Europe and temperate, tropical, arid and semi-arid Asia (Eurasia) and the Mediterranean region. It develops pink flowers from March to May in the younger appendages of the plant. Except available data on phenolic lipids such as flavonoids, tannins and lignans in Tamarix genus (Nawwar et al., 1982; Souliman et al., 1991; Parmar et al., 1994; Orabi et al., 2009) no other studies on chemical composition of T. canariensis have been reported previously in the literature. Phenolic compounds with linear alkyl chain attached to a single benzene ring are alkyl phenolics. When the nucleus to which the alkyl side-chain is attached is a dihydroxybenzene three isomers can exist. These structurally related compounds are called catechol, resorcinol and hydroquinone and they include two hydroxyl groups 2 linked to the aromatic ring in orto (1,2), meta (1,3) and para (1,4) positions, respectively. This group of molecules includes shortand long-chain members of the different isomeric series when they are alkylated with a frequently linear side-chain. 5-n-Alkylresorcinols (alkylresorcinols, ARs), also named 1,3-dihydroxy-5-alkylbenzene derivatives or cardols are long-chain dihydric alkylphenols more abundant than the alkylcatechols also called urushiols as natural compounds (Kozubek and Tyman, 1999). Both are also more abundant in nature than the alkyl derivatives of hydroquinone (Tyman, 1979). These amphiphilic phenolic compounds are recognized to be associated not only to higher plants but also with many other different organisms from bacteria and fungi to lower and higher plants (Kozubek and Tyman, 1999). Despite this heterogeneous origin they has been usually reported in high concentrations mainly in the outer layers of cereal (Gramineae ≡ Poaceae) grains specially in common wheat (Triticum aestivum L.), durum wheat (Triticum durum), rye (Secale cereale L.), triticale (Triticum x Secale) and barley (Hordeum vulgare L.). Minor amounts were isolated in some other higher plants used for food as in mango (Mangifera indica) (Knödler et al., 2009) and in lower plants like bacteria, mosses and algae, e.g: Apatococcus constipatus (Zarnowski et al., 2000). (S1) Supplementary material 1 shows as the homologue composition varies with species in which these compounds have been described in the literature and mainly as odd-numbered alkyl side-chain, the members of this class of compounds usually varies from C17 to C25 including minor amounts of unsaturated homologues (alk(en)ylresorcinols) with a chain unsaturation degree of one (mono-) or two (diunsaturated). Several epidemiological studies have associated the consumption of 3 foods as whole-grain cereals with decrease incidence of degenerative diseases including diabetes, obesity, coronary heart disease and some cancers (Ross et al., 2001, Chen et al., 2004). Figure 1 shows the molecular structure of the AR homologue with a saturated and straight hydrocarbon side-chain length of 21 carbon atoms and a single phenolic ring (5henicosyl-1,3-benzendiol). Congeners with an odd number of carbon atoms (C13-C27) that constitutes the alkyl side-chain attached to position 5 of the aromatic ring are most of times the major homologues described in higher plants. HO OH Figure 1. 5-n-heneicosylresorcinol (n-C21:0) molecular structure In almost the totality of cases the homologous series ranges from n-C17:0 to n-C25:0 being the maximum different within each species. Some of these include unsaturated members (alkenylresorcinols) but they are always in lower amounts as compared with the saturated counterparts. The aim of this study is to describe new natural products and their abundance in Tamarix through their chemical description, both qualitative and semiquantitative, of the main lipids extractable from the leave tissues of T. canariensis that were not previously described, to the best of our knowledge. As a consequence of the conducted analysis here is presented evidence of a new natural source of phenolic compounds which have been identified as 5-n-alkylguaiacols. We also found long-chain secondary alcohols and alkylresorcinols not previously characterized in Tamaricaceae. 4 2. Results and discussion 2.1. General aspects Although the total extract of the epicuticular waxes from the leaves of this higher plant contained several families of compounds some of them previously described by other authors in members of the genus Tamarix, we have focused our attention in the saltcedar composition on the three more abundant classes of chemical compounds: Alkylresorcinols, alkylguaiacols and secondary alcohols, all of them isolated for the first time from the saltcedar. Total contents of these three compounds classes were high, compared to those reported previously in other plant sources. Little is known about the content of alkyl phenolic derivatives in higher plants others than cereal species. Here in this study we present the results from the total lipid extraction of the leaves from the saltcedar. The TIC chromatogram (Figure 2) showed that the main peaks belonged to two series of novel phenolic compounds and eight homologous series of secondary alcohols. As Figure 2 shows, alkylphenolic derivatives are the main compounds in the overall chromatogram resulting from the total lipid extract of the leaves of the saltcedar. 5 Figure 2. Partial GC-MS Total Ion Current (TIC) from the silylated total extract of the leaves of Tamarix canariensis. Compounds were analyzed as TMS derivatives. IS = Internal standard. Cn = n-alkyl C number. Table 1 shows the content of ARs and alkylguaiacols (AGs) homologues as the sum of saturated derivatives with 13-27 carbon atoms in the side chain. The concentration of ARs was higher than reported by other studies excepting that reported by Landberg et al. (2008) on the intermediate layer of wheat (Triticum aestivum) grains (16,000 µg/g). Our results show also a homologue distribution very similar to the published results in 100 Relative abundance TMSO β-Sitosterol 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 Time (min) 0 10 20 30 40 50 60 70 80 90 TMSO OTMS 5-n-C 19 -AR 5-n-C 19 -AG 12-n-C 31 -ol 10-n-C31-ol 8-n-C31-ol n-C30-ol n-C28-ol O TMSO α-Tocopherol 5-n-C 21 -AR 5-n-C 21 -AG n-C30-al O OTMS TMSO n-C26-ol n-ω-C22-HA n-C29 n-C24-ol n-C32-al 5-n-C 22 -AG 5-n-C 17 -AR OTMS n-C22-ol O H OTMS TMSO OTMS 5-n-C 22 -AR 5-n-C 17 -AG TMSO OTMS OTMS OTMS OTMS OTMS OTMS OTMS TMSO OTMS O H OTMS n-C32-ol OCH3 TMSO OCH3 TMSO IS OCH 3 TMSO OCH 3 TMSO 6 other plant species. For example, Knödler et al. (2010) in a recent study developed in common wheat (Triticum aestivum) and durum wheat (T. durum) revealed in both cereal species that the homologues C21:0 and C19:0 were the most abundant. These series are nearly always dominated by the saturated homologues. However, Knödler et al. (2009) showed a profile dominated by two unsaturated homologues (C17:2 and C17:1) in mango (Mangifera indica) peel. Table 1. Alkylphenolic derivatives composition of cuticular waxes extracted from the leaves of Tamarix canariensis including their concentration and relative homologue abundance†, ǂ Alkylphenolic derivatives mg·kg-1 dry weight (%) Chain length # 5-n-alkylresorcinols 5-n-alkylguaiacols 13 not detected 1.4 (0.01) 14 7.0 (0.04) 2.8 (0.01) 15 235.1 (1.37) 192.3 (1.3) 16 9.8 (0.06) 7.8 (0.05) 17 1064.0 (6.18) 757.4 (5.2) 18 62.0 (0.36) 46.2 (0.3) 19 6089.0 (35.39) 4034.1 (27.9) 20 593.3 (3.45) 557.5 (3.9) 21 6313.3 (36.69) ‡ 5601.3 (38.8) 22 443.8 (2.58) 683.6 (4.7) 23 2035.2 (11.83) 2296.9 (16.0) 24 69.4 (0.40) 108.8 (0.7) 25 213.1 (1.24) 142.7 (1.0) 26 13.9 (0.08) 6.3 (0.04) 27 57.3 (0.33) 9.6 (0.07) Total 17,206 (100) 14,449 (100) †absolute values of individual homologues from each class of aliphatic compound are expressed in mg·kg-1 of dry weight (≡ µg·g-1 dw) ‡in bold are indicated the most abundant members of the series and in parenthesis their abundances ǂAbundance data obtained by peak integration from m/z 268 (ARs) and m/z 210 (AGs) mass chromatogram 2.2. 5-n-Alkylresorcinols (ARs) A homologous series of ARs with saturated alkyl side chain at position 5 of the benzene ring and varying from n-C13:0 to n-C27:0 with an odd over even dominance were identified in the total lipid extract from the leaves of the saltcedar. Total yield of these 7 phenolic lipids was 17.2 g·kg-1. The most abundant homologues were henicosylresorcinol (n-C21) (6.31 g/kg; 36.7%) and nonadecylresorcinol (n-C19) (6.09 g/kg; 35.4%). Despite this predominance of the odd members, the abundance of the even numbered alkyl chain homologues was not negligible. Figure 3 shows the mass chromatogram corresponding to the base peak at m/z 268 for the derivatized ARs. n-Alkylresorcinols are defined as phenolic lipids with an alkyl chain attached to position 5 of the 1,3-dihydroxybenzene ring. C 21 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 rt C 14 C 26 C 25 C 24 C 23 C 22 C 20 C 19 C 18 C 17 C 16 C 15 Figure 3. Mass chromatogram of base peak at m/z = 268 characteristic of the TMS derivatives of 5-n-alkylresorcinols extracted from leaves of the saltcedar (Tamarix canariensis). Cn = n-alkyl C number. 8 rearrangement and this ion occurs together with another at m/z 209 which is also prominent due to the dihydroxytropylium ion formed by direct β-cleavage (Deszcz and Kozubek, 2000; Ross et al., 2001), m/z 73 ([(CH3)3Si]+), m/z 223 which represents the result of a γ-cleavage with respect to the benzene ring. Finally, fragment ions m/z 195, m/z 210, m/z 223, m/z 252, m/z 447 and m/z 462 ([M]+) result from subtracting 58 Dalton (CH3)2Si to those of the corresponding 5-n-nonadecylresorcinol (Figure 4) providing unambiguous evidence on their molecular structure according to their alkyl chain length. Fragments m/z 121 and 135 are characteristic of guaiacols according to TTTT et al. (1999). The structural information for all AGs homologues described in this study is shown in Table 3. Table 3 Gas chromatographic – mass spectrometry structural characteristics of 5-n-alkylguaiacols as their TMS-ethers derivatives identified in the leaves from Tamarix canariensis. Compound R OCH 3 HO DB-5 retention time (min) [M]+ main fragments m/zǂ 1-Hydroxy-3-methoxy-5-n-tridecylguaiacol (n-C 13:0 ) 20,6 378(-) 210(-), 73(-), 223(-), 195(-), 252(-) 1-Hydroxy-3-methoxy-5-n-tetradecylguaiacol (n-C 14:0 ) 22,8 392(-) 210(-), 73(-), 223(-), 195(-), 252(-) 1-Hydroxy-3-methoxy-5-n-pentadecylguaiacol (n-C 15:0 ) 25,2 406(10) 210(100), 73(22), 223(14), 195(6), 252(4) 1-Hydroxy-3-methoxy-5-n-hexadecylguaiacol (n-C 16:0 ) 27,7 420(-) 210(-), 73(-), 223(-), 195(-), 252(-) 1-Hydroxy-3-methoxy-5-n-heptadecylguaiacol (n-C 17:0 ) 30,5 434(10) 210(100), 73(18), 223(14), 195(8), 252(6) 1-Hydroxy-3-methoxy-5-n-octadecylguaiacol (n-C 18:0 ) 33,2 448(-) 210(-), 73(-), 223(-), 195(-), 252(-) 1-Hydroxy-3-methoxy-5-n-nonadecylguaiacol (n-C 19:0 ) 36,2 462(32) 210(100), 73(48), 223(14), 195(8), 252(5) 1-Hydroxy-3-methoxy-5-n-eicosylguaiacol (n-C 20:0 ) 39,0 476(10) 210(100), 73(35), 223(41), 195(20), 252(15) 1-Hydroxy-3-methoxy-5-n-henicosylguaiacol (n-C 21:0 ) † 42,2 490(56) 210(100), 73(70), 223(68), 195(34), 252(28) 1-Hydroxy-3-methoxy-5-n-docosylguaiacol (n-C 22:0 ) 44,7 504(10) 210(100), 73(22), 223(12), 195(7), 252(4) 1-Hydroxy-3-methoxy-5-n-tricosylguaiacol (n-C 23:0 ) 47,6 518(14) 210(100), 73(29), 223(20), 195(12), 252(8) 1-Hydroxy-3-methoxy-5-n-tetracosylguaiacol (n-C 24:0 ) 50,8 532(10) 210(100), 73(35), 223(15), 195(8), 252(6) 1-Hydroxy-3-methoxy-5-n-pentacosylguaiacol (n-C 25:0 ) 54,6 546(10) 210(100), 73(-), 223(66), 195(8), 252(8) 1-Hydroxy-3-methoxy-5-n-hexacosylguaiacol (n-C 26:0 ) 59,2 560(-) 210(-), 73(-), 223(-), 195(-), 252(-) 1-Hydroxy-3-methoxy-5-n-heptacosylguaiacol (n-C 27:0 ) 64,7 574(-) 210(-), 73(-), 223(-), 195(-), 252(-) †In bold is indicated the homologue most abundant of this series of guaiacolic lipids ǂIons are ordered following the relative intensity to the base peak (100) (-) intensity not calculated due to their low abundance 15 Figure 6. Electron impact (EI) mass spectrum of the major 5-n-alkylguaiacol (1hydroxy-3-methoxy-5-n-nonadecylguaiacol ≡ 5-n-nonadecylguaiacol) (n-C19) present in the leaves of the saltcedar (Tamarix canariensis). The structure for that compound is also shown. 2.4. Asymmetrical n-secondary alcohols The extract of the leaves of the saltcedar is dominated by a secondary alkanol in the middle-right region of the TIC chromatogram (Fig. 1). Analysis of this abundant peak through its MS includes two prominent ions at m/z 257 (as the base peak) and m/z 369. These allowed us to identify it as the n-hentriacontan-12-ol (12-n-C31-ol). Having recognized all the members of this series of 12-fatty alcohols the others, namely 14-, 13-, 11-, 10-, 9-, 8and 7-hydroxyl fatty alcohols were also easily determined. So we present here for the first time in Tamarix genus to the best of our knowledge some of these homologous series of asymmetrical secondary alcohols. Table 4 resumes the 100 150 200 250 300 350 400 450 m/z 0 100 Relative Abundance 210 211 209 73 223 462 195 57 252 463 224 75 135 121 69 207 181 89 193 447 91 59 238 83 165 253 464 294 149 266 105 420 378 336 448 122 308 322 350 364 392 406 433 281 OCH 3 TMSO n-C19 m/z 210 M·+ [M-15]·+ 179 ·+ OCH 3 ·+ OCH3 ·+ ·+ + OCH 3 CH 2 H H TMSiO OCH 3 CH 2 H 2 C TMSiO + ·+ OCH 3 C C C CH 2 H 2 H 2 H 2 TMSiO CH 2 OCH 3 TMSiO Si CH 3 CH 3 H 3 C 16 reports on secondary alcohols isolated from members of the Tamaricaceae family conducted by several authors during the last decades and shows that they are apparently scarcely represented in this family. For example, Andhiwal and Kishore, 1984, identified almost exclusively the hentriacontan-12-ol homologue in Tamarix gallica. Different asymmetrical n-secondary alcohols cited in the literature of the Tamaricaceae family are described in Table 4. Table 4. Secondary alcohols previously identified in other studies on Tamaricaceae family Species Homologues detected Reference Myricaria paniculata - stems 12-n-C 31 -ol Li et al., 2005 Psorospermum androsaemifolium - leaves 12-n-C 31 -ol Poumale et al., 2008 Tamarix dioica 7-n-C 31 -ol Agrawal et al., 1991 Tamarix gallica – Aerial parts 10-n-C 29 -ol, 12-n-C 29 -ol, 12-n-C 31 -ol (97%), 12-n-C 33 -ol, 14-n-C 33 -ol Andhiwal and Kishore, 1984 Tamarix hampeana - flowers 19-n-C 46 -ol Aykaç and Akgül, 2010 For diagnostic purposes Figure 7 shows how the relative position of the hydroxyl group over the aliphatic chain of these compounds allows the unambiguous identification of the homologue of each series (from 7to 14-) due to the formation of two very distinctive ions. For example, the branched alcohol at the C-12 position resulting from the n-hentriacontane paraffin as their trimethylsilylated derivative show the following two prominent fragments due to the α-cleavage, m/z 257, which is the diagnostic ion for all members of the 12series and m/z 369. Corresponding to the other α-cleavage. OTMS OTMS OTMS Hentriacontan-12-ol Hentriacontan-11-ol Hentriacontan-10-ol OTMS OTMS OTMS Hentriacontan-9-ol Hentriacontan-8-ol Hentriacontan-7-ol m/z 257 m/z 369 m/z 397 m/z 229 m/z 425 m/z 201 m/z 411 m/z 215 m/z 243 m/z 283 m/z 187 m/z 439 17 Figure 7. Main fragments observed in the mass spectra of the asymmetrical secondary alcohols identified in the leaves of the saltcedar with the hydroxyl group located in the 12-, 11-, 10-, 9-, 8and 7positions when the homologues are derived of the hentriacontan-X-ol where X represents the carbon atom which supports the functional group. Figure 8 shows the concentration of the 53 homologues expressed as mg·kg-1 of dry weight of these series of n-secondary alcohols identified in our study evidencing that the n-C33 member was the most abundant among the 14and 13-n-fatty alcohols, n-C31 member was the most abundant among the 12119and 8-n-fatty alcohols while the nC29 was clearly the major in the 10and 7series. The most abundant isomer homologue was hentriacontan-12-ol. Finally, the n-alkanes (n-C19 – n-C32) of the leaves of this plant maximized together with several of these fatty alcohols at odd carbon numbered homologues (C-29 and C-31). These could suggest that they are synthesized by a different pathway from those homologues such as the n-alkanols (n-C7 – n-C33) (Cmax = n-C30) and n-alkanoic acids (n-C6 – n-C33) (Cmax = n-C16 and n-C30). 0 50 100 150 200 250 300 350 400 450 25 26 27 28 29 30 31 32 33 34 35 TConcentration (mg/kg dry weight) 0 10 20 30 40 50 60 70 80 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 0 5000 10000 15000 20000 25000 30000 35000 40000 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 0 20 40 60 80 100 120 140 160 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 14-n-Fatty alcohols 13-n-Fatty alcohols 12-n-Fatty alcohols 11-n-Fatty alcohols 0 500 1000 1500 2000 2500 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 0 1 2 3 4 5 6 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 0 50 100 150 200 250 300 350 400 450 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 0 5 10 15 20 25 30 25 26 27 28 29 30 31 32 33 34 35 Concentration (mg/kg dry weight) 10-n-Fatty alcohols 9-n-Fatty alcohols 8-n-Fatty alcohols 7-n-Fatty alcohols 18 Figure 8. Homologue series of all 53 secondary alcohols (14-, 13-, 12-, 11-, 10-, 9-, 8and 7-) identified in the leaves of the saltcedar (Tamarix canariensis). Below (Figure 9) is presented the mass spectrum of the hentriacontan-12-ol as its trimethylsilylated derivative. The fragmentation was dominated as was indicated before by two prominent ions in the middle of the MS resulting from the α-cleavage to the TMSi group and both fragments enable to locate the position of the OH group of the original alcohol as was indicated in a previous study on the secondary alcohols from several plants (Holloway et al., 1976). So, in this n-C31 homologue TMSi ether derivative mass spectrum, ion m/z 257, that is also the base ionic peak, corrresponding to a fragment [C12H24OSi(CH3)3]·+ derived from the shorter chain end resulting from α-cleavage, was slightly stronger (and the base peak) than the second α-fragment m/z 369 [C20H40OSi(CH3)3]·+ from the longer chain end. This sylilated derivative was further characterized by the molecular ion m/z 524 which shows very low abundancy, the M-15 fragment m/z 509 due to the loss of a methyl group and the expected sylilated fragments derived from fatty alcohols m/z 75 (ca 75% rel. intensity) > 103 (ca 69% rel. intensity) altogether with the trimethylsilyl (TMS) fragment ion (m/z = 73) (ca 79% rel. intensity) which was the most abundant of these ions in the lower end of the MS. 19 Figure 9. Electron impact (EI) mass spectrum n-hentriacontan-12-ol identified in the leaves of the saltcedar (Tamarix canariensis) and representative of the respective homologous series of asymmetrical n-secondary alcohols. The structure for that compound is also shown. 3. Conclusions The qualitative composition and semiquantitative content of three groups of natural compounds investigated in the saltcedar (Tamarix canariensis Willd.) revealed that could be an important source of naturally occurring non-isoprenoid polyketideeederived phenols both 5-n-alkylresorcinols and 5-n-alkylguaiacols. Here in this study, it has been detected: i. Two homologous series of phenolic lipids, namely 1,3-dihydroxy-5-alkylbenzenes (alkylresorcinols, ARs) and 1-hydroxy-3methoxy-5-n-alkylguaiacol (alkylguaiacols, AGs) had very similar pattern and characterized by substituent alkyl chains of length C13 to C27 and including the even50 100 150 200 250 300 350 400 450 500 550 m/z 0 10 20 30 40 50 60 70 80 90 100 257 369 73 75 258 103 83 370 57 129 69 111 85 259 371 115 143 509 241 185 260 510 229 372 171 157 199 283 353 397 297 327 524 434 425 444 493 474 589 574 547 n-C31 M·+ m/z 369 m/z 257 [M-15]+ OTMS 97 20 numbered members were found to be present in the total lipid extract of the leaves from the saltcedar though the odd carbon number homologues dominated. A homologous series of 5-n-alkylguaiacols described for the first time as natural compounds is reported here with a mass spectrometric and gas chromatographic behavior similar to that of ARs. Although odd-numbered hydrocarbon side chain homologues of the ARs and AGs were more abundant members of the series; however, even numbers of carbon atoms were also present. Finally, 53 compounds comprising eight homologous series of secondary alcohols (14-, 13-, 12-, 11-, 10-, 9-, 8and 7-) centered at the n-C31 homologue were also described in Tamarix for the first time. 4. Materials and methods 4.1. Sampling site Branches from the saltcedar (Tamarix canariensis) were collected near the Font Salada (46,19º N; 4,8º E; 332 m) in the county of Bages (Barcelona, Catalonia) close to an hypersaline well in the Salat creek near where this tributary of the Gavarresa stream finishes. Samples were transported to the laboratory and further analyzed. A voucher specimen (BCN 106840) was deposited at the Herbarium of the Faculty of Pharmacy, Universitat de Barcelona. 4.2. Analytical procedures 4.2.1. GC/MS preanalytical conditions: sample treatment, extraction, derivatization Fresh leaves were air-dried at room temperature and crushed and homogenized in a glass mortar using a glass pestle using 25g of previously cleaned sea sand. All inert materials and tools were previously cleaned and rinsed with solvents before use. Ground 21 samples were introduced in cellulose thimbles and extracted in a Söxhlet apparatus during 24 h using a mixture of the organic solvents pentane / dichloromethane (DCM) (7:3, v/v). In our investigation the extraction of 7,1 g of milled leaves was performed in darkness conditions to avoid photo-oxidation processes of the compounds. 300 µL (0,06 mg) of internal standard (Friedeline; Aldrich) (200 mg·L-1) were added to the extract which was subsequently rotoevaporated to approximately 0,5 mL and further concentrated under a nitrogen (N2) stream. The extract of the sample was derivatized prior to performing the GC/MS analysis. Silylation of hydroxyl and carboxyl groups to get TMSi-ethers and esters respectively was achieved by addition of 300 µL N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA; Merck) and heating at 70ºC for 1h prior GC-MS analysis. 4.2.2. GC/MS analysis One microlitre of derivatized sample was injected (275ºC) in a Fisons Instruments Gas Chromatograph equipment in splitless mode coupled to a mass detector (GC 8000/MD 800). Separation was performed on a DB-5ms column (30-m long by 0,32 mm i.d. fused silica capillary column coated with a 0,25 µm low-polarity liquid phase film thickness of 5% methylpolysiloxane, J&W Scientific Folsom, California). The mass scanning in total ion count (TIC) was acquired in the range of 50-650 daltons over 1s. The oven temperature was programmed as follows: starting at 40ºC for 1 minute, and up to 230ºC at a rate of 20ºC min-1, then from 230ºC to 300ºC at 2ºC min-1 with a 20 min holding time. Helium was used as the mobile carrier gas with a flow rate of 1,0 mL·min-1. The inlet and transfer line temperatures were set at 300 and 310ºC respectively. The ion source temperature was 250ºC and ionization was in electronic impact mode (70 eV). Ion source and interface temperatures were 200 and 300ºC respectively. 22 4.3. Identification of the compounds Compounds were identified by chromatographic and spectroscopic means. In this study ARs compounds were identified by comparing their characteristics mass fragmentation patterns and retention times with those reported in literature (Avsejs et al., 2002; Ross et al., 2004). The identification of the new 5-n-alkylguaiacols (AGs) homologues was deduced similarly with the ARs from the molecular ion and the base peak m/z 210. 4.4. Quantification The relative compositions and total amounts of the homologues were estimated from the integrated area of the peaks in the TIC using the MassLab software package. The semiquantitative results were obtained by using the single internal standard, in our case friedeline. 5. References Agrawal., P.K., 1991. Hentriacontan-7-ol and hentriacont-7Z-ene from Tamarix dioica. Planta Med. 57, 397. Andhiwal, C.K., Kishore, K., 1984. Hentriacontan-12-ol and other waxy constituents of Tamarix gallica Linn. J. Indian Chem. Soc. 61, 820-822. Athukorala, Y., Hosseinian, F.S., Mazza, G., 2010. Extraction and fractionation of alkylresorcinols from triticale bran by two-step supercritical carbon dioxide. LWT - Food Sci. Technol. 43, 660-665. 23 Avsejs, L.A., Nott, Ch.J., Xie, Sh., Maddy D., Chambers, F.M., Evershed, R.P., 2002. 5-n-Alkylresorcinols as biomarkers of sedges in an ombrotrophic peat section. Organic Geochemistry 33, 861-867. Aykaç A., Akgül Y., 2010. A new analogue of fatty alcohol from Tamarix hampeana L. Nat. Prod. Res. 24, 34-39. Billets, S., Craig, J.C., Corbett, M.D., Vickery, J.F., 1976. Component analysis of the urushiol content of poison ivy and poison oak. Phytochemistry 15, 533-535. Briggs, D.E., 1974. Hydrocarbons, phenols and sterols of the testa and pigment strand in the grain of Hordeum distichon. Phytochemistry 13, 987-996. Brown, G.D., 1992. Two new compounds from Artemisia annua. Journal of Natural Products 55, 1756-1760. Chen, Y., Ross, A.B., Åman, P., Kamal-Eldin, A., 2004. Alkylresorcinols as markers of whole grain wheat and rye in cereal products. J. Agr. Food Chem. 52, 8242-8246. Deszcz, L., Kozubek, A., 2000. Higher cardol homologs (5-alkylresorcinols) in rye seedlings. Biochim. Biophys. Acta 1483, 241-250. Effendi, A., Gerhauser, H., Bridgewater, A.V., 2008. Production of renewable phenolic resins by thermochemical conversion of biomass: A review. Renewable and sustainable energy Reviews 12, 2092-2116. 24 S1 (Continued) Origin, source, plant tissue, concentration (µg/g), Homologue composition† and name of the constituents Reference Triticum aestivum (Soft wheat): n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (489-1.429). Ross et al., 2003 Triticum aestivum (Soft wheat): bran oil, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (5-n-(14’-(Z)-nonadecenyl)resorcinol, 5-n-(2’-keto-14’-(Z)- heneicosenyl)resorcinol, 5-n-(2’-ketoheneicosyl)resorcinol), (19100). Iwatsuki et al., 2003 Triticum aestivum (soft winter & spring wheat): grains, n-C 15:0 , n-C 17:1 , n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:0 , n-C 23:1 , n-C 23:0 , n-C 25:0 , (212-240). Zarnowski et al., 2004 Triticum aestivum (Common spring wheat): grains, n-C 19:1 (x2), n-C 17:0 , n-C 19:1 (x3), n-C 19:0 , n-C 21:1 (x2), n-C 21:0 , n-C 23:0 , n-C 25:0 , (19-302). Zarnowski and Suzuki., 2004 Triticum aestivum (Soft wheat): spring and winter varieties (227 – 639): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 and cereal ingredients: whole grain wheat, rye and barley – rye bran and flour – wheat bran (412/726/8 – 2.758/99 – 2.211): n-C17:0, n-C19:0, n-C21:0, n-C23:0, n-C25:0 Chen et al., 2004 Triticum aestivum (Soft wheat): crisp bread, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (308) Linko-Parvinen et al., 2007 Triticum sp. (Soft wheat): grains, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 ; whole grain wheat intact and milled (576 and 528); wheat bran and aleurone milled (2.923 and 1.024). Landberg et al., 2007 Triticum aestivum (Soft wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , winter wheat grain (220-652) and spring wheat grain (254-537). Andersson et al., 2008 Triticum aestivum (Soft wheat): whole-grain flour, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , 47; whole grain pasta, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C23:0, n-C25:0, 194; refined grain crisp bread, n-C17:0, n-C19:0, n-C21:0, n-C23:0, n-C25:0, 31; refined grain soft bread, n-C17:0, n-C19:0, n-C21:0, n-C23:0, n-C25:0, 37; refined grain flour, n-C17:0, n-C19:0, n-C21:0, n-C23:0, n-C25:0; 25; refined grain pasta, n-C19:0, n-C21:0, n-C23:0, n-C25:0; 8. Landberg et al., 2008 Triticum aestivum (Common spring wheat): n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:1 , n-C 21:0 , n-C 23:1 , n-C 23:0 , n-C 25:0 ; outer parts, kernels, of grains (500-655). Landberg et al., 2009 Triticum aestivum (Winter wheat): n-C 17:1 , n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:1 , n-C 21:0 , n-C 23:1 , n-C 23:0 , n-C 25:1 , n-C 25:0 ; outer parts, kernels, of grains (494-536). Landberg et al., 2009 Triticum aestivum (Soft wheat): whole grains (740-758), ground grains (725-740). Sampietro et al., 2009 Triticum aestivum (Soft wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , wholegrain flour (490-710), refined flour (36), bran (3625), wholegrain spelt wheat flour (650) refined spelt wheat flour (80). Andersson et al., 2010 Triticum sp. (Soft wheat): bran, n-C 15:0 , n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:1 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (680). Athukorala et al., 2010 Triticum aestivum (Soft wheat): whole wheat grains (763-971), ground wheat grains (658-748), whole wheat bran (2759-3186), whole wheat flour (243-269). Gajda et al., 2008 Triticum sp. (Soft wheat): bran, n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:1 , n-C 23:oxo , n-C 21:0 , n-C 23:0 , (2672-3645). Liu et al., 2012 Triticum aestivum (Soft wheat): bran, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (1365). Holt et al., 2012 Triticum aestivum (Soft wheat): bread whole grain, (241-677). Shewry et al., 2013 Triticum aestivum subsp. spelta (Spelt), (490-741). Shewry et al., 2013 Triticum spp. (Wheat): n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25: 0; whole-wheat flour (759), white flour (47), organic white flour (44) and bran (3225). Mattila et al., 2005 Triticum dicoccum, (531-714). Shewry et al., 2013 Triticum durum (Hard wheat): grains: n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:1 , n-C 21:0 , n-C 23:0 , n-C 25:0 . Hengtrakul et al., 1991 Triticum durum (Hard wheat): grains, n-C 15:0 , n-C 17:0 , n-C 19:1 , n-C 19:0 , n-C 21:0 , n-C 23:1 , n-C 23:0 , n-C 25:0 ; (185-238). Zarnowski et al., 2004a Triticum durum (Hard wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , kernels (251-618) and pasta products (215-270). Landberg et al., 2006 Triticum durum (Hard wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (194-531). Andersson et al., 2008 Triticum durum (Hard wheat): outer parts, kernels, of grains, n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (370-452). Landberg et al., 2009 Triticum durum (Hard wheat): (194-531) Shewry et al., 2013 Triticum monococum (Einkorn): (540-654). Shewry et al., 2013 Triticum spelta (Spelt wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (490-741). Andersson et al., 2008 Triticum spelta (Einkorn wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (545-654). Andersson et al., 2008 Triticum spelta (Emmer wheat): n-C 17:0 , n-C 19:0 , n-C 21:0 , n-C 23:0 , n-C 25:0 , (531-714). Andersson et al., 2008 Zea mays (Maize): whole grains (10-13) and ground grains (10-12). Sampietro et al., 2009 †in bold is indicated the most abundant homologue, when data is available ‡:0 denotes carbon numbered and number of insaturations of n-alkyl substituent 31 S2. Histogram of 5-n-alkylresorcinols contained in the leaves of the saltcedar (Tamarix canariensis). Numbers in abscissas refer to the chain length. 0 1000 2000 3000 4000 5000 6000 7000 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Concentration (mg/kg dry weight) S3. Histogram of 5-n-alkylguaiacols contained in the leaves of the saltcedar (Tamarix canariensis)…………………………………………………………………………….S3 0 1000 2000 3000 4000 5000 6000 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Concentration (mg/kg dry weight) S4. EI mass spectra and structural information for 5-n-alkyl-m-guaiacols extracted from the leaves of T. canariensis including the following evenand odd-numbered hydrocarbon side chains homologues: n-C15:0 (A), n-C17:0 (B), n-C19:0 (C), n-C20:0 (D), n-C21:0 (E), n-C22:0 (F), n-C23:0 (G), n-C24:0 (H) and n-C25:0 (I) represented by the molecular ion peaks at 406, 434, 462, 476, 490, 504, 518 and 532 m/z, respectively. Structure for the n-C21:0, the most abundant homologue, is also shown. 32 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 73 55 57 209 195 223 406 391 n-C15:0 (A) [M]+ = 406 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 73 57 209 195 223 434 252 419 n-C 17:0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 209 73 57 195 135 121 89 91 223 462 252 447 n-C 19:0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 73 57 209 195 223 297 252 354 323 476 n-C 20:0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 209 73 57 195 135 121 181 223 490 252 238 475 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 73 57 209 195 223 504 252 489 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 209 73 57 195 75 223 518 252 503 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 73 57 195 223 281 268 252 532 517 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 m/z 0 100 % 210 223 195 252 73 57 546 531 n-C 25:0 (B) [M]+ = 434 (D) [M]+ = 476 (C) [M]+ = 462 (E) [M]+ = 490 n-C 21:0 n-C 22:0 n-C 24:0 n-C 23:0 (F) [M]+ = 504 (G) [M]+ = 518 (I) [M]+ = 546 OCH 3 TMSO (H) [M]+ = 532 210 33 To date the study of the fragmentation of compounds with the base peak at m/z 210 has not been made because the natural occurrence of these compounds has also not been documented in the literature. 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